The aim of this grant proposal is to understand the role of the thymus and thymic trafficking dendritic cells (DCs) in oral tolerance. We have shown that oral administration of antigen (Ag) can affect central tolerance through mechanisms of clonal deletion of developing Ag-specific thymocytes, or by induction of natural regulatory T cells (nTregs), depending on the model Ag used. We present preliminary evidence that specialized gut-derived DC populations access intestinal Ags and transport them to the thymus, which is critical to central tolerance induction by oral Ags. We therefore propose that oral Ags are sampled by gut- associated DCs and transported via the blood into the thymus, where they can impact central tolerance of developing thymocytes specific to oral Ags. Studies under Aim 1 will characterize gut-associated peripheral DC populations that traffic to the thymus homeostatically, and after Ag feeding, and will determine the effects of defined Ag peptides and of gut-associated DC subsets on the developmental fate of Ag-specific thymocytes in vivo (i.e. clonal deletion vs. nTreg induction). Studies under Aim 2 will establish the role of endogenous DC subsets and their thymic trafficking programs on central tolerance to oral Ags. Studies under Aim 3 will explore the requirements of defined endogenous DC subsets and their thymic trafficking programs on the prevention of autoimmunity in oral tolerance. More importantly they will establish a critical role for dominant thymic selection events (i.e. thymic nTreg induction) after oral Ag delivery, in controlling autoimmunity during oral tolerance, because this outcome of thymic tolerance generates immunosuppressive cells with the ability to regulate peripheral T cell responses. Therefore oral tolerance protocols need to be revisited in the clinic with the aim of possibly targeting younger individuals with a higher thymic T cell output or using Ags that elicit the development of immunosuppressive thymic T cell populations vs. the clonal deletion of Ag-specific thymocytes.